Alzheimer""s disease (AD), first described by the Bavarian psychiatrist Alois Alzheimer in 1907, is a progressive neurological disorder that begins with short term memory loss and proceeds to disorientation, impairment of judgment and reasoning and, ultimately, dementia. The course of the disease usually leads to death in a severely debilitated, immobile state between four and 12 years after onset. AD has been estimated to afflict 5 to 11 percent of the population over age 65 and as much as 47 percent of the population over age 85. The societal cost for managing AD is upwards of 80 billion dollars annually, primarily due to the extensive custodial care required for AD patients. Moreover, as adults born during the population boom of the 1940""s and 1950""s approach the age when AD becomes more prevalent, the control and treatment of AD will become an even more significant health care problem. Currently, there is no treatment that significantly retards the progression of the disease. For reviews on AD, see Selkoe, D. J. Sci. Amer. , November 1991, pp. 68-78; and Yankner, B. A. et al. (1991) N. Eng. J. Med. 325:1849-1857.
It has recently been reported (Games et al. (1995) Nature 373:523-527) that an Alzheimer-type neuropathology has been created in transgenic mice. The transgenic mice express high levels of human mutant amyloid precursor protein and progressively develop many of the pathological conditions associated with AD.
Pathologically, AD is characterized by the presence of distinctive lesions in the victim""s brain. These brain lesions include abnormal intracellular filaments called neurofibrillary tangles (NTFs) and extracellular deposits of amyloidogenic proteins in senile, or amyloid, plaques. Amyloid deposits are also present in the walls of cerebral blood vessels of AD patients. The major protein constituent of amyloid plaques has been identified as a 4 kilodalton peptide called xcex2-amyloid peptide (xcex2-AP)(Glenner, G. G. and Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 120:885-890; Masters, C. et al. (1985) Proc. Natl. Acad. Sci. USA 82:4245-4249). Diffuse deposits of xcex2-AP are frequently observed in normal adult brains, whereas AD brain tissue is characterized by more compacted, dense-core xcex2-amyloid plaques. (See e.g., Davies, L. et al. (1988) Neurology 38:1688-1693) These observations suggest that xcex2-AP deposition precedes, and contributes to, the destruction of neurons that occurs in AD. In further support of a direct pathogenic role for xcex2-AP, xcex2-amyloid has been shown to be toxic to mature neurons, both in culture and in vivo. Yankner, B. A.et al. (1989) Science 245:417-420; Yankner, B. A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9020-9023; Roher, A. E. et al. (1991) Biochem. Biophys. Res. Commun. 174:572-579; Kowall, N. W. et al. (1991) Proc. Natl. Acad. Sci. USA 88:7247-7251. Furthermore, patients with hereditary cerebral hemorrhage with amyloidosis-Dutch-type (HCHWA-D), which is characterized by diffuse xcex2-amyloid deposits within the cerebral cortex and cerebrovasculature, have been shown to have a point mutation that leads to an amino acid substitution within xcex2-AP. Levy, E. et al. (1990) Science 248:1124-1126. This observation demonstrates that a specific alteration of the P-AP sequence can cause xcex2-amyloid to be deposited.
Natural xcex2-AP is derived by proteolysis from a much larger protein called the amyloid precursor protein (APP). Kang, J. et al. (1987) Nature 325:733; Goldgaber, D. et al. (1987) Science 235:877; Robakis, N. K. et al. (1987) Proc. Natl Acad Sci. USA 84:4190; Tanzi, R. E. et al. (1987) Science 235:880. The APP gene maps to chromosome 21, thereby providing an explanation for the xcex2-amyloid deposition seen at an early age in individuals with Down""s syndrome, which is caused by trisomy of chromosome 21. Mann, D. M. et al. (1989) Neuropathol. Appl. Neurobiol. 15:317; Rumble, B. et al. (1989) N. Eng. J. Med. 320:1446. APP contains a single membrane spanning domain, with a long amino terminal region (about two-thirds of the protein) extending into the extracellular environment and a shorter carboxy-terminal region projecting into the cytoplasm. Differential splicing of the APP messenger RNA leads to at least five forms of APP, composed of either 563 amino acids (APP-563), 695 amino acids (APP-695), 714 amino acids (APP-714), 751 amino acids (APP-751) or 770 amino acids (APP-770).
Within APP, naturally-occurring xcex2 amyloid peptide begins at an aspartic acid residue at amino acid position 672 of APP-770. Naturally-occurring xcex2-AP derived from proteolysis of APP is 39 to 43 amino acid residues in length, depending on the carboxy-terminal end point, which exhibits heterogeneity. The predominant circulating form of xcex2-AP in the blood and cerebrospinal fluid of both AD patients and normal adults is xcex21-40 (xe2x80x9cshort xcex2xe2x80x9d). Seubert, P. et al. (1992) Nature 359:325; Shoji, M. et al. (1992) Science 258:126. However, xcex21-42 and xcex21-43 (xe2x80x9clong xcex2xe2x80x9d) also are forms in xcex2-amyloid plaques. Masters, C. et al. (1985) Proc. Natl. Acad. Sci. USA 82:4245; Miller, D. et al. (1993) Arch. Biochem. Biophys. 301:41; Mori, H. et al. (1992) J. Biol. Chem. 267:1 7082. Although the precise molecular mechanism leading to xcex2-APP aggregation and deposition is unknown, the process has been likened to that of nucleation-dependent polymerizations, such as protein crystallization, microtubule formation and actin polymerization. See e.g., Jarrett, J. T. and Lansbury, P. T. (1993) Cell 73:1055-1058. In such processes, polymerization of monomer components does not occur until nucleus formation. Thus, these processes are characterized by a lag time before aggregation occurs, followed by rapid polymerization after nucleation. Nucleation can be accelerated by the addition of a xe2x80x9cseedxe2x80x9d or preformed nucleus, which results in rapid polymerization. The long xcex2 forms of xcex2-AP have been shown to act as seeds, thereby accelerating polymerization of both long and short xcex2-AP forms. Jarrett, J. T. et al. (1993) Biochemistry 32:4693.
In one study, in which amino acid substitutions were made in xcex2-AP, two mutant xcex2 peptides were reported to interfere with polymerization of non-mutated xcex2-AP when the mutant and non-mutant forms of peptide were mixed. Hilbich, C. et al. (1992) J. Mol. Biol. 228:460-473. Equimolar amounts of the mutant and non-mutant (i.e., natural) xcex2 amyloid peptides were used to see this effect and the mutant peptides were reported to be unsuitable for use in vivo. Hilbich, C. et al. (1992), supra.
This invention pertains to compounds, and pharmaceutical compositions thereof, that can bind to natural xcex2 amyloid peptides (xcex2-AP), modulate the aggregation of natural xcex2-AP and/or inhibit the neurotoxicity of natural xcex2-APs. The compounds are modified in a manner which allows for increased biostability and prolonged elevated plasma levels. The xcex2-amyloid modulator compounds of the invention comprise a peptidic structure, preferably based on xcex2-amyloid peptide, that is composed entirely of D-amino acids. In various embodiments, the peptidic structure of the modulator compound comprises a D-amino acid sequence corresponding to a L-amino acid sequence found within natural xcex2-AP, a D-amino acid sequence which is an inverso isomer of an L-amino acid sequence found within natural xcex2-AP, a D-amino acid sequence which is a retro-inverso isomer of an L-amino acid sequence found within natural xcex2-AP, or a D-amino acid sequence that is a scrambled or substituted version of an L-amino acid sequence found within natural xcex2-AP. Preferably, the D-amino acid peptidic structure of the modulator is designed based upon a subregion of natural xcex2-AP at positions 17-21 (Axcex217-20 and Axcex217-21, respectively), which has the amino acid sequences Leu-Val-Phe-Phe-Ala (SEQ ID NO:4). In preferred embodiments, a phenylalanine in the compounds of the invention is substituted with a phenylalanine analogue which is more stable and less prone to, for example, oxidative metabolism, or allows for increased brain levels of the compound.
In yet another embodiment, a modulator compound of the invention includes a xcex2-amyloid peptide comprised of D-amino acids, L-amino acids or both, an inverso isomer of a xcex2-amyloid peptide, or a retro-inverso isomer of a xcex2-amyloid peptide which is attached to a hydrazine moiety, wherein the compound binds to natural xcex2-amyloid peptides or modulates the aggregation or inhibits the neurotoxicity of natural xcex2-amyloid peptides when contacted with the natural xcex2-amyloid peptides.
A modulator compound of the invention preferably comprises 3-20 D-amino acids, more preferably 3-10 D-amino acids and even more preferably 3-5 D-amino acids. The D-amino acid peptidic structure of the modulator can have free amino-, carboxy-, or carboxy amide-termini Alternatively, the amino-terminus, the carboxy-terminus or both may be modified. For example, an N-terminal modifying group can be used that enhances the ability of the compound to inhibit AP aggregation. Moreover, the amino- and/or carboxy termini of the peptide can be modified to alter a pharmacokinetic property of the compound (such as stability, bioavailability, e.g., enhanced delivery of the compound across the blood brain barrier and entry into the brain, and the like). Preferred amino-terminal modifying groups include alkyl groups, e.g., methyl, ethyl, or isopropyl groups. Preferred carboxy-terminal modifying groups include amide groups, alkyl or aryl amide groups (e.g., phenethylamide), hydroxy groups (i.e., reduction products of peptide acids, resulting in peptide alcohols), acyl amide groups, and acetyl groups. Still further, a modulator compound can be modified to label the compound with a detectable substance (e.g., a radioactive label).
In certain preferred embodiments, the invention provides a compound having the structure: N,N-dimethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N,N-dimethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-methyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-ethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-isopropyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-isopropylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-dimethylamide; N,N-diethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N,N-diethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N-dimethyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; H-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-ethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-ethyl-(Gly-D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-methyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-ethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-propyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N-diethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ile)-NH2; H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ala-)-NH2; H-(D-Ile-D-Ile-D-Phe-D-Phe-D-Ile)-NH2; H-(D-Nle-D-Val-D-Phe-D-Phe-D-Ala-)-NH2; H-(D-Nle-D-Val-D-Phe-D-Phe-D-Nle)-NH2; 1-piperidine-acetyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; 1-piperidine-acetyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; H-D-Leu-D-Val-D-Phe-D-Phe-D-Leu-isopropylamide; H-D-Leu-D-Phe-D-Phe-D-Val-D-Leu-isopropylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-methylamide; H-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-methylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-OH; N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-[F5]Phe-D-Leu)-NH2; H-(D-Leu-D-Phe-D-Cha-D-Val-D-Leu)-NH2; H-(D-Leu-D-Phe-D-[p-F]Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Phe-D-[F5]Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Phe-D-Lys-D-Val-D-Leu)-NH2; H-(D-Leu-D-Cha-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-[p-F]Phe-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-[F5]Phe-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Lys-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Cha-D-Cha-D-Val-D-Leu)-NH2; H-(D-Leu-D-[p-F]Phe-D-[p-F]Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-[F5]Phe-D-[F5]Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Lys-D-Lys-D-Val-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe-D-[F5]Phe-D-Leu)-NH2; H-D-Leu-D-Val-D-Phe-NH-(H-D-Leu-D-Val-D-Phe-)NH; H-D-Leu-D-Val-D-Phe-NHxe2x80x94NHxe2x80x94COCH3; and H-D-Leu-D-Val-D-Phe-NHxe2x80x94NH2.
Particularly preferred compounds of the invention are set forth in the Examples.
Another aspect of the invention pertains to pharmaceutical compositions. Typically, the pharmaceutical composition comprises a therapeutically effective amount of a modulator compound of the invention and a pharmaceutically acceptable carrier.
Yet another aspect of the invention pertains to methods for inhibiting aggregation of natural xcex2-amyloid peptides. These methods comprise contacting the natural xcex2-amyloid peptides with a modulator compound of the invention such that aggregation of the natural xcex2-amyloid peptides is inhibited.
Yet another aspect of the invention pertains to methods for detecting the presence or absence of natural xcex2-amyloid peptides in a biological sample. These methods comprise contacting a biological sample with a compound of the invention, wherein the compound is labeled with a detectable substance, and detecting the compound bound to natural xcex2-amyloid peptides to thereby detect the presence or absence of natural xcex2-amyloid peptides in the biological sample.
Still another aspect of the invention pertains to methods for treating a subject for a disorder associated with xcex2-amyloidosis. These methods comprise administering to the subject a therapeutically effective amount of a modulator compound of the invention such that the subject is treated for a disorder associated with xcex2-amyloidosis. Preferably, the disorder is Alzheimer""s disease. Use of the modulators of the invention for therapy or for the manufacture of a medicament for the treatment of a disorder associated with xcex2-amyloidosis is also encompassed by the invention.